The present invention concerns a cloned human neurokinin-3 receptor (hereinafter identified as human NK3R).
Neurokinin B (NKB) is a naturally occuring peptide belonging to the neurokinin family of peptides, which also includes substance P (SP) and substance K (SK). NKB binds preferentially to the neurokinin-3 receptor (NK3R), although it also recognizes the other two receptor subtypes (NK1 and NK2) with lower affinity. As is well known in the art, neurokinin B and other tachykinins have been implicated in the pathophysiology of numerous diseases. Neurokinin peptides are reportedly involved in nociception and neurogenic inflammation. The physiological function of NK3R has been implicated in the regulation of enkephalin release, while the NK1 and NK2 receptor subtypes are involved in synaptic transmission (Laneuville et al., Life Sci., 42:1295-1305 (1988)). Since the NKB genomic structure and subcellular distribution are different from those of SP and SK, the physiological function and regulatory mechanism of NKB may be different from SP and SK.
More specifically, neurokinin B is a pharmacologically-active neuropeptide that is produced in mammals and possesses a characteristic amino acid sequence that is illustrated below:
Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2.
Several groups have reported the cloning of certain neurokinin receptors. T. M. Fong, et al., Mol. Pharmacol., 41:24-30 (1991) have reported cloned human neurokinin-1 and neurokinin-1 short form receptor. J. Yokota, et al., J. Biol. Chem., 264:17649 (1989) have reported cloned rat neurokinin-1 receptor. N. P. Gerard, et al., J. Biol. Chem., 265:20455 (1990), have reported human neurokinin-2 receptor. Cloned rat and bovine neurokinin-2 receptor have likewise been reported. See respectively, Y. Sasi, and S. Nakanishi, Biochem Biophys. Res. Comm., 165:695 (1989), and Y. Masu, et al., Nature 329:836 (1987). Cloned rat neurokinin-3 receptor has been reported by R. Shigemoto, et al., J. Biol. Chem., 265:623 (1990). The above references, however, neither disclose nor suggest the present invention.
The instant invention also concerns an assay protocol which can be used to determine the activity in body fluids of substances that bind human NK3R; these include neurokinin B. The assay can also be used for identifying and evaluating substances that bind NK3R. Thus, the assay can be used to identify neurokinin B antagonists and evaluate their binding affinity. Another method for an assay includes that described by M. A. Cascieri, et al., J. Biol. Chem., 258:5158 (1983). See also, for example, R. M. Snider, et al., Science, 251:435 (1991) and S. McLean, et al., Science, 251:437 (1991). See also WIPO Patent Publications WO90/05525 and WO90/05729, published May 31, 1990. Methods to date have proven inferior, in part, for failure of the animal receptor (animal NK1R, NK2R or NK3R) activity to accurately reflect that of the human neurokinin-3 receptor. Furthermore, prior to this disclosure, human NK3R has not been available in a highly purified form or in substantial isolation from NK1R and/or NK2R. Use of such neurokinin receptor sources can not accurately depict the affinity of an agonist or an antagonist for a human NK3R.
A novel recombinant human neurokinin-3 receptor (hereinafter identified as human NK3R) is disclosed which has been prepared by polymerase chain reaction techniques. Also disclosed is the complete sequence of human NK3R complementary DNA; expression systems, including a CHO (chinese hamster ovarian cell line) stable expression system; and an assay using the CHO expression system.
Human NK3R can be used in an assay to identify and evaluate entities that bind neurokinin B receptor or NK3R. The assay can also be used in conjunction with diagnosis and therapy to determine the body fluid concentration of neurokinin-B related substances in patients. In addition, the complete sequence of the human NK3R is useful in the process of developing novel NK3 agonists and antagonists by computer modeling.
One embodiment of the invention concerns human neurokinin-3 receptor, said receptor being free of other human receptor proteins.
In one class this first embodiment concerns human neurokinin-3 receptor, said receptor being free of other human proteins.
Within this class, this first embodiment concerns human neurokinin-3 receptor from human cells such as glioblastoma, said receptor being free of other human proteins.
Also within this class, this first embodiment concerns human neurokinin-3 receptor, the receptor being recombinantly produced from non-human cells.
In a second class, this first embodiment concerns a protein corresponding to the amino acid sequence of human neurokinin-3 receptor, the protein comprising 465 amino acids. Within the second class this first embodiment concerns a protein comprising the following 465 amino acid sequence (SEQ ID NO:1:) depicted from the amino to the carboxy terminus:
Within the second class this first embodiment also concerns a protein comprising the foregoing amino acid sequence (SEQ ID:NO:1:), the protein being free of other human receptor proteins.
A second embodiment concerns a DNA sequence encoding the human neurokinin-3 receptor, the DNA sequence being free of other human DNA sequences.
As will be appreciated by those of skill in the art, there is a substantial amount of redundancy in the set of codons which translate specific amino acids. Accordingly, the invention also includes alternative base sequences wherein a codon (or codons) are replaced with another codon, such that the amino acid sequence translated by the DNA sequence remains unchanged. For purposes of this specification, a sequence bearing one or more such replaced codons will be defined as a degenerate variation. Also included are mutations (exchange of individual amino acids) which one of skill in the art would expect to have no effect on functionality, such as valine for leucine, arginine for lysine and asparagine for glutamine.
One class of the second embodiment of the invention concerns the following nucleotide sequence (SEQ ID NO:2:) of complementary DNA depicted from the 5xe2x80x2 to the 3xe2x80x2 terminus:
or a degenerate variation thereof.
A third embodiment of this invention concerns systems for expressing all or part of the human neurokinin-3 receptor.
One class this third embodiment of the invention comprises:
A plasmid which comprises:
(a) a mammalian expression vector, such as pcDNAI/Neo, and
(b) a base sequence encoding human neurokinin-3 receptor protein.
Within this first class of the third embodiment the neurokinin-3 receptor comprises the nucleotide sequence (SEQ ID NO:2:) of complementary DNA as shown above.
A second class of this third embodiment of the invention concerns a system for the transient expression of human neurokinin-3 receptor in a monkey kidney cell line (COS), the system comprised of a vector which expresses human neurokinin receptor (human NK3R) cDNA.
Within this second class of the third embodiment is the sub-class wherein the expression system includes:
A plasmid which comprises:
(a) a mammalian expression vector, such as pcDNAI/Neo, and
(b) a base sequence encoding human neurokinin-3 receptor protein.
A third class of this third embodiment of the invention concerns a system for the expression of human neurokinin-3 receptor in a chinese hamster ovarian cell line (CHO), the system comprising a vector comprising human neurokinin-3 receptor (human NK3R) cDNA.
Within this third class of the third embodiment is the sub-class wherein the expression system includes:
A plasmid which comprises:
(a) a mammalian expression vector, such as pcNDAI/Neo and
(b) a base sequence encoding human neurokinin-3 receptor protein.
Within this sub-class the neurokinin-3 receptor expression system comprises the nucleotide sequence (SEQ ID NO:2:) of complementary DNA as shown above.
It is understood, and is readily apparent to those skilled in the art that a wide variety of commonly used cell lines are suitable for use in the present invention. Suitable cell lines derived from various species include, but are not limited to, cell lines of human, bovine, porcine, monkey, and rodent origin, or from yeast and bacterial strains.
A fourth embodiment of the invention concerns a method of using any of the above expression systems for determining the binding affinity of a test sample for human neurokinin-3 receptor.
In one class this fourth embodiment concerns a method of using a Chinese hamster ovarian cell line (CHO), the line transplanted with a plasmid,
which plasmid comprises:
(a) a mammalian expression vector, such as pcDNAI/Neo, and
(b) a base sequence encoding human neurokinin-3 receptor protein,
the method which comprises:
(1) expressing human neurokinin-3 receptor in the CHO cells;
(2) adding of a test sample to a solution containing 125I-eledoisin and the CHO cells;
(3) incubating the products of Step (2), the incubation being effective for competitive binding of the 125I-eledoisin and said test sample to the human neurokinin-3 receptor;
(4) separating the 125I-eledoisin which is bound to the human neurokinin-3 receptor from the 125I-eledoisin which is not bound;
(5) measuring the amount of the 125I-eledoisin which is bound to the human neurokinin-3 receptor.
In a second class this fourth embodiment concerns a method of using a monkey kidney cell line (COS), the line transplanted with a plasmid,.
which plasmid comprises:
(a) a mammalian expression vector, such as pcDNAI/Neo, and
(b) a base sequence encoding human neurokinin-3 receptor protein,
the method which comprises:
(1) expressing human neurokinin-3 receptor in the COS cells;
(2) adding of a test sample to a solution containing 125I-eledoisin and the COS cells;
(3) incubating the products of Step (2), the incubation being effective for competitive binding of the 125I-eledoisin and said test sample to the human neurokinin-3 receptor;
(4) separating the 125I-eledoisin which is bound to the human neurokinin-3 receptor from the 125I-eledoisin which is not bound;
(5) measuring the amount of the 125I-eledoisin which is bound to the human neurokinin-3 receptor.
In a third class this fourth embodiment concerns a method of using a Chinese hamster ovarian cell line (CHO), the line transplanted with a plasmid,
which plasmid comprises:
(a) a mammalian expression vector, such as pcDNAI/Neo, and
(b) the base sequence encoding human neurokinin-3 receptor protein,
the method which comprises:
(1) expressing human neurokinin-3 receptor in the CHO cells;
(2) equilibrating the product of Step (1) with 3H-myoinositol;
(3) washing the product of Step (2);
(4) incubating the product of Step (3) with a test sample and neurokinin-B in the presence of aqueous LiCl, resulting in the production of 3H-inositol monophosphate;
(5) measuring the 3H-inositol monophosphate.
In overview, the present invention describes methods to isolate the human neurokinin-3 receptor (human NK3R) complementary DNA (cDNA) without prior knowledge of its protein sequence or gene sequence. A polymerase chain reaction (PCR) technique was utilized for the isolation of human NK3R cDNA. In the approach, the regions of rat NK3R sequence thought to be similar to human NK3R were identified, oligonucleotide primers corresponding to those region were designed, PCR amplification was carried out to obtain a partial clone of the NK3R cDNA from human cells, and its DNA sequence was determined. The full length cDNA encoding the human NK3R was obtained from human mRNA utilizing the previous sequence information.
The complete sequence of the human NK3R cDNA was determined, and its encoded protein sequence was deduced. Among other things, such sequence information is useful in the process of developing novel neurokinin B antagonists.
Three heterologous expression systems were developed to express the cloned human NK3R cDNA. The Xenopus oocyte expression enables one to determine the biological function of human NK3R. The COS (a monkey kidney cell line) expression can be used to measure the ligand binding properties of human NK3R. The CHO (a Chinese hamster ovarian cell line) stable expression is suitable for natural product screen to identify potential therapeutic agents or other substances that bind to neurokinin-3 receptor or human NK3R. The cell line can also be used for determining the concentration of neurokinin B in human samples.
Assay protocols were developed to use the heterologously expressed human NK3R for the determination of the binding affinity and efficacy of neurokinin B agonists/antagonists with therapeutic potential.